Team:Peking S/lab/notebook/lhc

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== '''summary''' ==
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== '''Summary''' ==
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Hanchi lin, who also names himself as Edward in English, is a 3rd year undergraduate majoring in bioscience. He joined the Peking_S IGEM team with the intention of broadening his vision of synthetic biology. Through the whole summer he has been doing experiment and making efforts to promote our project procedure. When occupied in nothing, he loves to playing basketball, watching movies and running.
+
My major task is molecule cloning of generators and corresponding receivers for chemical wires such as A-factor. In addition, characterization of some logic gates like AND GATE as well as constructing a three input gate using new signaling molecules is my major contribution in the later stage of our poject. What is more, I am also responsible for the direction and manufacture of the CG animation about invertors.
=='''Contents'''==
=='''Contents'''==
Line 83: Line 83:
===7.1===
===7.1===
-
1.  the PCR primers is delivered and get the product: arac+Pbad+supD+nahr+Psal by PCR.
+
1.  Learn the basic experiment skills including transformation and ligation and digestion..
-
2.  double digestion of the PCR product.
+
===7.2===
===7.2===
1.  Electrophoresis enzyme digestion reaction system is a 1.5% agarose gel.
1.  Electrophoresis enzyme digestion reaction system is a 1.5% agarose gel.
 +
2.  Excise the gel slice and extract the DNA fragments.
2.  Excise the gel slice and extract the DNA fragments.
 +
3.  Ligation of insert DNA fragments for 3 hours.
3.  Ligation of insert DNA fragments for 3 hours.
 +
4.  Transform the products of ligation.
4.  Transform the products of ligation.
===7.3===
===7.3===
-
1.  DNA double digestion of the plasmid including Pbad.
+
1.  DNA double digestion of the plasmid including pbad.
-
2.  Positively transform the part 1-14N.
+
 
-
3.  Ligation of Pbad and gfp.
+
2.  Positively transform the part 1-14N(2011).
 +
 
 +
3.  Ligation of pbad and gfp.
===7.4===
===7.4===
-
1.  Transform the products of ligation of Pbad and GFP and cultivate the plate overnight.
+
1.  Transform the products of ligation of pbad and gfp and cultivate the plate overnight.
===7.5===
===7.5===
1.  pick five clones cultivated overnight on the plate.
1.  pick five clones cultivated overnight on the plate.
 +
2.  Using the clones to PCR to verify the DNA fragments are connected correctly.
2.  Using the clones to PCR to verify the DNA fragments are connected correctly.
 +
3.  And cultivate the bacteria in LB for 10 hours.
3.  And cultivate the bacteria in LB for 10 hours.
 +
4.  According the result of PCR, choose the correct cloning to miniprep the plasminds.
4.  According the result of PCR, choose the correct cloning to miniprep the plasminds.
===7.6===
===7.6===
-
1.  Get the ligation of GFP and nahr+Psal+GFP transformed.
+
1.  Get the ligation of gfp and nahr+psal+gfp transformed.
 +
 
2.  Pick up the clones and cultivate the bacteria overnight.
2.  Pick up the clones and cultivate the bacteria overnight.
Line 118: Line 126:
1.  Get the plasmid coding for “Pbad+GFP+Psal+GFP”.
1.  Get the plasmid coding for “Pbad+GFP+Psal+GFP”.
 +
2.  DNA double digestion of part 1-5G(Xbal and Pstl) and part 1-7C(Xbal and Pstl).
2.  DNA double digestion of part 1-5G(Xbal and Pstl) and part 1-7C(Xbal and Pstl).
 +
3.  DNA double digestion of plasmid coding for “Pt7+GFP” and plasmid coding for “T7ptag”.
3.  DNA double digestion of plasmid coding for “Pt7+GFP” and plasmid coding for “T7ptag”.
Line 124: Line 134:
1.  Ligation of T7ptag and terminator.
1.  Ligation of T7ptag and terminator.
 +
2.  Transform the ligation product.
2.  Transform the ligation product.
-
3.  After pick some clones to pcr, cultivate the same clones.
+
 
-
4.  Successfully get the plasmid Pbad+GFP+Psal+GFP, which can severs as an or gate.
+
3.  After pick some clones to PCR, cultivate the same clones.
 +
 
 +
4.  Successfully get the plasmid coding for"Pbad+GFP+Psal+GFP", which can operate as an demonstration of OR GATE.
===7.15===
===7.15===
1.  Find the accurately ligation to miniprep.
1.  Find the accurately ligation to miniprep.
-
2.  Try to link the supD+terminator before Pt7+GFP+terminator, fail.
+
 
-
3.  Spread the bacteria with plasmid Pbad+GFP+Psal+GFP on four different plates with salicylic acid, arabinose, both of two and none of two.
+
2.  Try to link the supD+terminator before pt7+GFP+terminator, fail.
 +
 
 +
3.  Spread the bacteria with plasmid pbad+gfp+psal+gfp on four different plates with salicylic acid, arabinose, both of two and neither of two.
===7.16===
===7.16===
-
1.  Success ligation of the supD+terminator in the stern of arac+Pbad+supD+nahr+Psal.
+
1.  Success ligation of the supD+terminator in the downstream of arac+pbad+supD+nahr+psal.
-
2.  Cut the backbones including 3T5,4K5.
+
 
 +
2.  Cut the backbones including psb3T5,psb4K5.
===7.17===
===7.17===
-
1.  Positively transform the plasmid from parts 1-15P.
+
1.  Positively transform the plasmid from part 1-15P.
-
2.  Try to link the rbs+arac+Pbad+supD+nahr+Psal+supD with Pt7+GFP+terminator, fail.
+
 
 +
2.  Try to link the rbs+arac+pbad+supD+nahr+psal+supD with pt7+gfp+terminator, fail.
===7.18===
===7.18===
-
1.  Find out that the both rbs+arac+Pbad+supD+nahr+Psal+supD and Pt7+GFP+terminator have a long sequence,thus it is difficult to connect them in a usual way.
+
1.  Find out that the both rbs+arac+pbad+supD+nahr+psal+supD and Pt7+gfp+terminator have a long sequence,thus it is difficult to connect them in a usual way.
===7.19 - 7.20===
===7.19 - 7.20===
-
1.  We decide to use a method called ”three pieces connected”.
+
1.  We decide to use a method called ”three pieces jointed”.
-
2.  Get the plasmid rbs+arac+Pbad+supD+nahr+Psal+supD double digested (Ecorl Spel) and plasmid Pt7+GFP+terminator double digested(Xbal Pstl).
+
 
-
3.  Connect the two DNA fragments with 3T5.
+
2.  Get the plasmid rbs+arac+pbad+supD+nahr+psal+supD double digested (Ecorl Spel) and plasmid pt7+gfp+terminator double digested(Xbal Pstl).
-
4.  Get the newly gained plasmid coding for Pqrr by PCR and get it double digested.
+
 
 +
3.  Connect the two DNA fragments with psb3T5.
 +
 
 +
4.  Get the newly gained plasmid coding for pqrr by PCR and get it double digested.
===7.21===
===7.21===
-
1.  Miniprep the Pqrr+T7ptag+terminator and double digest it to switch another backbone.
+
1.  Miniprep the pqrr+T7ptag+terminator and double digest it to switch another backbone.
===7.26 - 7.27===
===7.26 - 7.27===
-
1.  Find the concentration of rbs+arac+Pbad+supD+nahr+Psal+supD+Pt7+GFP+terminator which has been sent for sequencing is too low to sequencing.
+
1.  Find the concentration of rbs+arac+pbad+supD+nahr+psal+supD+pt7+gfp+terminator which has been sent for sequencing is too low to sequencing.
 +
 
2.  Pick the clones and we deliver the plasmid for sequencing once more.
2.  Pick the clones and we deliver the plasmid for sequencing once more.
===7.28 - 7.30===
===7.28 - 7.30===
-
1.  Find that Pqrr+T7ptag+terminator has two rbs, so we start to PCR the T7ptag again.
+
1.  Find that pqrr+T7ptag+terminator has two ribosome binding sites, so we start to PCR the T7ptag again after figuring out what happen.
==August==
==August==
Line 224: Line 245:
1.  Since the sequencing result has come, we are sure that we has got our plasmids validly assembled.
1.  Since the sequencing result has come, we are sure that we has got our plasmids validly assembled.
 +
2.  Plasmids including:
2.  Plasmids including:
-
Pqrr+T7ptag+terminator (rbs: b0034);.
+
pqrr+T7ptag+terminator (rbs: b0034);.
-
Pqrr+T7ptag+terminator (rbs: b0035);
+
pqrr+T7ptag+terminator (rbs: b0035);
===8.2 - 8.3===
===8.2 - 8.3===
-
1.  Send the rbs+arac+Pbad+supD+nahr+Psal+supD+Pt7+GFP+terminator plasmid for sequencing.
+
1.  Send the rbs+arac+pbad+supD+nahr+psal+supD+pt7+gfp+terminator plasmid for sequencing.
-
2.  Connect the Pqrr+Cqsa.
+
 
 +
2.  Connect the pqrr+cqsa.
===8.4===
===8.4===
1.  Ultimately, get all the obligable plasmid clones assembled correctly.
1.  Ultimately, get all the obligable plasmid clones assembled correctly.
 +
2.  Switch the plasmid to corresponding backbone.  
2.  Switch the plasmid to corresponding backbone.  
Line 241: Line 265:
1.  Assist in the induction of XOR gate.
1.  Assist in the induction of XOR gate.
-
2.  The primary result are not satisfying, hence, start to have the rbs strength mutation.
+
 
 +
2.  The primary result are not satisfying, hence, start to have the rbs strength mutated.
==September==
==September==
Line 304: Line 329:
Use PlacI invertor to construct a NOT GATE.
Use PlacI invertor to construct a NOT GATE.
 +
Not mainly in charge of some specific mission, help others to promot the progress of our project.
Not mainly in charge of some specific mission, help others to promot the progress of our project.

Latest revision as of 00:17, 6 October 2011


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Hanchi Lin's Notebook


Summary

My major task is molecule cloning of generators and corresponding receivers for chemical wires such as A-factor. In addition, characterization of some logic gates like AND GATE as well as constructing a three input gate using new signaling molecules is my major contribution in the later stage of our poject. What is more, I am also responsible for the direction and manufacture of the CG animation about invertors.

Contents

July

Mon Tue Wed Thu Fri Sat Sun
- - - - 1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
- - - - - - -

[TOP]

7.1

1. Learn the basic experiment skills including transformation and ligation and digestion..

7.2

1. Electrophoresis enzyme digestion reaction system is a 1.5% agarose gel.

2. Excise the gel slice and extract the DNA fragments.

3. Ligation of insert DNA fragments for 3 hours.

4. Transform the products of ligation.

7.3

1. DNA double digestion of the plasmid including pbad.

2. Positively transform the part 1-14N(2011).

3. Ligation of pbad and gfp.

7.4

1. Transform the products of ligation of pbad and gfp and cultivate the plate overnight.

7.5

1. pick five clones cultivated overnight on the plate.

2. Using the clones to PCR to verify the DNA fragments are connected correctly.

3. And cultivate the bacteria in LB for 10 hours.

4. According the result of PCR, choose the correct cloning to miniprep the plasminds.

7.6

1. Get the ligation of gfp and nahr+psal+gfp transformed.

2. Pick up the clones and cultivate the bacteria overnight.

7.7 - 7.10

1. Get the plasmid coding for “Pbad+GFP+Psal+GFP”.

2. DNA double digestion of part 1-5G(Xbal and Pstl) and part 1-7C(Xbal and Pstl).

3. DNA double digestion of plasmid coding for “Pt7+GFP” and plasmid coding for “T7ptag”.

7.14

1. Ligation of T7ptag and terminator.

2. Transform the ligation product.

3. After pick some clones to PCR, cultivate the same clones.

4. Successfully get the plasmid coding for"Pbad+GFP+Psal+GFP", which can operate as an demonstration of OR GATE.

7.15

1. Find the accurately ligation to miniprep.

2. Try to link the supD+terminator before pt7+GFP+terminator, fail.

3. Spread the bacteria with plasmid pbad+gfp+psal+gfp on four different plates with salicylic acid, arabinose, both of two and neither of two.

7.16

1. Success ligation of the supD+terminator in the downstream of arac+pbad+supD+nahr+psal.

2. Cut the backbones including psb3T5,psb4K5.

7.17

1. Positively transform the plasmid from part 1-15P.

2. Try to link the rbs+arac+pbad+supD+nahr+psal+supD with pt7+gfp+terminator, fail.

7.18

1. Find out that the both rbs+arac+pbad+supD+nahr+psal+supD and Pt7+gfp+terminator have a long sequence,thus it is difficult to connect them in a usual way.

7.19 - 7.20

1. We decide to use a method called ”three pieces jointed”.

2. Get the plasmid rbs+arac+pbad+supD+nahr+psal+supD double digested (Ecorl Spel) and plasmid pt7+gfp+terminator double digested(Xbal Pstl).

3. Connect the two DNA fragments with psb3T5.

4. Get the newly gained plasmid coding for pqrr by PCR and get it double digested.

7.21

1. Miniprep the pqrr+T7ptag+terminator and double digest it to switch another backbone.

7.26 - 7.27

1. Find the concentration of rbs+arac+pbad+supD+nahr+psal+supD+pt7+gfp+terminator which has been sent for sequencing is too low to sequencing.

2. Pick the clones and we deliver the plasmid for sequencing once more.

7.28 - 7.30

1. Find that pqrr+T7ptag+terminator has two ribosome binding sites, so we start to PCR the T7ptag again after figuring out what happen.

August

Mon Tue Wed Thu Fri Sat Sun
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31 - - - -

[TOP]

8.1

1. Since the sequencing result has come, we are sure that we has got our plasmids validly assembled.

2. Plasmids including: pqrr+T7ptag+terminator (rbs: b0034);. pqrr+T7ptag+terminator (rbs: b0035);

8.2 - 8.3

1. Send the rbs+arac+pbad+supD+nahr+psal+supD+pt7+gfp+terminator plasmid for sequencing.

2. Connect the pqrr+cqsa.

8.4

1. Ultimately, get all the obligable plasmid clones assembled correctly.

2. Switch the plasmid to corresponding backbone.

8.5 - 8.20

1. Assist in the induction of XOR gate.

2. The primary result are not satisfying, hence, start to have the rbs strength mutated.

September

Mon Tue Wed Thu Fri Sat Sun
- - 1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 - --

[TOP]

9.1 - 9.5

1． Use other promoters to substitute the original promoter for construct other AND GATE.

9.6 - 9.30

Use PlacI invertor to construct a NOT GATE.

Not mainly in charge of some specific mission, help others to promot the progress of our project.

October

Mon Tue Wed Thu Fri Sat Sun
- - - - 1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 - - - - - -

[TOP]

10.1

10.3

10.4

10.5

10.7

10.8

10.9

10.10

10.11

10.12

10.13

10.15

10.16-10.21

10.21-10.25

 

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